The focus of this work has been to understand the molecular details that control initial steps in the recognition of cells infected with pathogens such as viruses by cells of the innate and adaptive immune systems. Understanding the function, mechanism, structure, and evolution of the interaction of virus-encoded molecules recognized by the immune system can lead not only to a deeper understanding of molecular interactions in general and of cell-cell interactions in the immune system, but also may lead to rational approaches to intervention in virus infection and neoplasia. In particular, we study representative members of the large family of major histocompatibility complex (MHC)-encoded molecules from a biophysical and structural perspective. We are interested in how MHC-I molecules interact with receptors on natural killer (NK) cells and on T lymphocytes through their NK and T cell receptors, respectively. Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy, and we have directed our efforts to understand the function, cellular expression, and structure of a set of these MHC-I (referred to as MHC-Iv) molecules encoded by the mouse cytomegalovirus (mCMV). We have analyzed the expression of several of these genes after transfection in different cell types, and have established that, unlike the classical MHC-I molecules, the viral MHC-I molecules do not require either the light chain component of the classical MHC-I molecule, beta-2 microglobulin, or self-peptide for expression. Although several of these MHC-Iv molecules are expressed at the surface of virus-infected cells early after infection, several others, including m152 and m155 are not expressed well at the cell surface, suggesting that their functions result from intracellular activities. In earlier studies, we determined the structures of the MHC-Iv molecules, m144, m152, and m153. Each of these molecules represents a different mode of immunoevsive action. m152 down-regulates host molecules crucial for recognition by either T cells or NK cells -- specifically it down-regulates host MHC-I molecules to elude CD8 T cell recognition. In addition, m152 down-regulates ligands of the NKG2D NK cell activating receptor, in particular the RAE-1 family of stress induced molecules. m153 has an unknown function, but is highly conserved in sequence among a number of mCMV strains that derive from wild mice, suggesting a conserved function. Our studies of m152 demonstrated the direct interaction of this mCMV encoded MHC-Iv protein both with host MHC-I and the stress-induced RAE-1 molecules, and we have determined the X-ray crystallographic structure of m152 in complex with its RAE-1 ligand. This structure reveals a novel adaptation of the MHC-I protein fold for binding to RAE-1, another member of the MHC-I family. Since the NK activating receptor, NKG2D also interacts with RAE-1, it was important to compare the interaction of m152 with RAE-1 with the interaction of NKG2D. Surprisingly, the sites of interaction are the same, and competition experiments confirm this. The details of the interaction of m152 with RAE-1 have been confirmed by examination of the binding of some 18 site directed mutants of RAE-1. Thus m152 provides a novel example of an mCMV-encoded MHC-I-like protein that binds two different classes of MHC-I-like proteins. The mCMV protein m153 provides another unique example of the varied functions of mCMV MHC-Iv molecules. Although the precise function of m153 is not known, we have explored this extensively using a reporter cell system, in which m153 expressing cells become fluorescent on ligation of their surface expressed m153 by cells bearing an m153 ligand. Our studies of the function of m153 have been complemented by the determination of the crystallographic structure of this molecule, which has been solved and refined to 2.4 Angstrom resolution. The most striking feature of this new MHC-like structure is that the molecule forms a stable head to tail homodimer. To confirm the dimerization interface observed in the crystal structure of m153 we have analyzed a number of interface mutants, confirming the site of dimerization. In addition to the m145 family of mouse CMV molecules, we have directed considerable attention to another novel family of putative immunoevasins of the MCMV, the m04 family. These molecules, that include m02, m04, and m06 seem to have distinct functions. m04 accompanies the MHC-I molecule to the cell surface, and m06 directs MHC-I molecules to an endosomal/lysomal pathway. This is a distinct protein family, and efforts to explore the structure of any of its members crystallographically have been difficult. We have successfully engineered m04 and examined its binding to MHC-I to quantify this interaction. We have determined the structure of m04 in solution using NMR in collaboration with Drs.Nik Sgourakis and Ad Bax. NMR structure determination is based on determination of multidimensional spectra that allow assignment of molecular restraints to various interatomic distances within the molecule. Because we expressed the recombinant molecule in bacteria, we were able to label it with a variety of isotopic precursors (13C, 15N, 2D, in various combinations) permitting gathering of spectra allowing interpretation of different interatomic distances. To reduce the amount of data needed, allowing us to employ sparse NMR data, Drs. Sgourakis and Bax exploited a new technology using Rosetta modeling to determine the solution structure of the m04 protein. This structure is a unique beta-fold, based distantly on the Ig-fold, that is representative of the full m02-m06 family of viral proteins. Thus, we not only determined a completely new structure in solution, indicative of a previously elusive protein family, but have applied a novel methodology was recently published in Structure. We have extended these findings on m04 to the related MCMV immunoevasin, m06. This molecule has been engineered for expression in E. coli, and binding studies indicated that it interacts with MHC-I molecules with low but detectable affinity. Using a recombinant MHC-I molecule that gives an outstanding multidimensional NMR spectrum, we have recently been able to map the MHC-I binding site of m06. This work has recently been published.

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9
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2016
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Natarajan, Kannan; Jiang, Jiansheng; May, Nathan A et al. (2018) The Role of Molecular Flexibility in Antigen Presentation and T Cell Receptor-Mediated Signaling. Front Immunol 9:1657
Sgourakis, Nikolaos G; May, Nathan A; Boyd, Lisa F et al. (2015) A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR. J Biol Chem 290:28857-68
Sgourakis, Nikolaos G; Natarajan, Kannan; Ying, Jinfa et al. (2014) The structure of mouse cytomegalovirus m04 protein obtained from sparse NMR data reveals a conserved fold of the m02-m06 viral immune modulator family. Structure 22:1263-1273
Wang, Rui; Natarajan, Kannan; Revilleza, Maria Jamela R et al. (2012) Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1? reveals a paradigm for MHC/MHC interaction in immune evasion. Proc Natl Acad Sci U S A 109:E3578-87
Revilleza, Maria Jamela; Wang, Rui; Mans, Janet et al. (2011) How the virus outsmarts the host: function and structure of cytomegalovirus MHC-I-like molecules in the evasion of natural killer cell surveillance. J Biomed Biotechnol 2011:724607
Zhi, Li; Mans, Janet; Paskow, Michael J et al. (2010) Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms. Biochemistry 49:2443-53
Mans, Janet; Zhi, Li; Revilleza, Maria Jamela R et al. (2009) Structure and function of murine cytomegalovirus MHC-I-like molecules: how the virus turned the host defense to its advantage. Immunol Res 43:264-79
Margulies, David H (2009) Home schooling of NK cells. Immunity 30:313-5